Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds
Metabolomic profile and nucleoside composition of Cordyceps nidus sp. nov. (Cordycipitaceae): A new source of active compounds
Juan ChirivÂõ 0 1
Giovanna Danies 0 1
Rocio Sierra 1
Nicolas Schauer 1
Sandra Trenkamp 1
Silvia Restrepo 0 1
Tatiana Sanjuan 1
☯ These authors contributed equally to this work. 1
0 Laboratory of Mycology and Plant Diseases, Universidad de los Andes, Bogot aÂ, Colombia, 2 Product and Processes Design Group, Universidad de los Andes , Bogot aÂ, Colombia, 3 Metabolomic Discoveries GmbH, Postdam-Golm, Germany , 4 Laboratorio de TaxonomÂõa y EcologÂõa de Hongos, Universidad de Antioquia , MedellÂõn , Colombia
1 Editor: Sabrina Sarrocco, Universita degli Studi di Pisa , ITALY
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported in part from the
project ªThe phylogenetic relationship of
Cordyceps in the Insecta Class in the Amazon of
Colombiaº Code 489-2009 (COLCIENCIAS,
Department of Science and Technology of
Colombia), and the project MetaboloÂmics de
Cordyceps nidus and Cordyceps takaomontana: a
Cordyceps sensu lato is a genus of arthropod-pathogenic fungi, which have been used
traditionally as medicinal in Asia. Within the genus, Ophiocordyceps sinensis is the most coveted
and expensive species in China. Nevertheless, harvesting wild specimens has become a
challenge given that natural populations of the fungus are decreasing and because
largescale culture of it has not yet been achieved. The worldwide demand for products derived
from cultivable fungal species with medicinal properties has increased recently. In this
study, we propose a new species, Cordyceps nidus, which parasitizes underground nests of
trapdoor spiders. This species is phylogenetically related to Cordyceps militaris, Cordyceps
pruinosa, and a sibling species of Cordyceps caloceroides. It is found in tropical rainforests
from Bolivia, Brazil, Colombia and Ecuador. We also investigated the medicinal potential of
this fungus based on its biochemical properties when grown on four different culture media.
The metabolic profile particularly that of nucleosides, in polar and non-polar extracts was
determined by UPLC, and then correlated to their antimicrobial activity and total phenolic
content. The metabolome showed a high and significant dependency on the substrate used
for fungal growth. The mass intensities of nucleosides and derivative compounds were
higher in natural culture media in comparison to artificial culture media. Among these
compounds, cordycepin was the predominant, showing the potential use of this species as an
alternative to O. sinensis. Furthermore, methanol fractions showed antimicrobial activity
against gram-positive bacteria, and less than 3.00 mg of gallic acid equivalents per g of
dried extract were obtained when assessing its total phenolic content by modified
Folin-Ciocalteu method. The presence of polyphenols opens the possibility of further exploring the
antioxidant capacity and the conditions that may enhance this characteristic. The metabolic
composition and biochemical activity indicate potential use of C. nidus in pharmaceutical
bioprospective analysis, code 1204-669-46686
(COLCIENCIAS) to metabolomics analysis and to
prepare and publish the manuscript. Metabolomic
Discoveries GmbH provides support in the form of
salaries for authors NS and ST but did not have any
additional role in the study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. The specific roles of these authors
are articulated in the ªauthor contributions" section.
Competing interests: Nicolas Schauer and Sandra
Trenkamp are employed by Metabolomic
Discoveries GmbH. There are no patents, products
in development or marketed products to declare.
This does not alter our adherence to all the PLOS
ONE policies on sharing data and materials, as
detailed online in the guide for authors.
Consumer demand for natural products is growing worldwide. Fungi represent an important
source of novel bioactive entities for drug development and therapeutic agents [
ascomycete Cordyceps sensu lato (s.l.) is a large genus of arthropod-pathogenic fungi located in the
order Hypocreales, some of which are traditionally used as medicinal mushrooms in China.
Among these, the lepidopteran pathogen Ophiocordyceps sinensis (syn.Cordyceps sinensis) is
perhaps the most widely renowned species with pharmaceutical properties [
]. This fungus is
only found buried deep in the ground parasitizing larvae of the ghost moth Thitarodes spp
(Lepidoptera: Hepialide) up to 5.000 meters of altitude in the eastern and southern parts of the
Tibetan Plateau [
]. However, its supply is rapidly decreasing mainly due to its
]. Hirsutella sinensis is currently recognized as its asexual state in cultivable species,
based on the observation of its microcyclic conidiation and molecular analyses [
Unfortunately, its large-scale cultivation has not yet been achieved due to the fungus' strong host
]. Thus, other species such as Cordyceps militaris and Cordyceps pruinosa which
parasitize many genera of lepidopteran larvae have received further attention because they are
generalist and have a similar metabolic composition as O. sinensis [4, 7±9].
The medicinal properties of Cordyceps s.l. have been mainly attributed to nucleosides,
glycosylamines involved in the regulation and modulation of several biological processes and
their derivatives [10±11]. These may be extracted from fruiting bodies or mycelia and are
currently studied as therapeutic agents [7±8, 10, 12±13]. Nucleosides can be differentially induced
by intrinsic or environmental factors, such as the developmental stage of the fungus, light
exposure and wavelength, the composition of the culture media, among other factors [7±8],
[12±15]. Traditionally, the nucleosides cordycepin (3'-deoxyadenosine), adenosine, inosine,
guanosine, and uridine have been used as markers to assess the quality of natural and cultured
species of Cordyceps [4, 16±17].
In this study, we propose a new species of Cordyceps that is pathogen of trapdoor spiders
collected from tropical rainforest in the Neotropics. The metabolomic profile of this new
taxon was also explored through the assessment of the intracellular extracts in mycelial
growing on different culture media. The analysis focused in the nucleosides compounds and
nonreported metabolites. Additionally, we evaluated the medicinal potential of this species by
measuring antimicrobial activity and total phenolic content.
Materials and methods
Collections were made in the first expedition under research permit number Res: 0871±2014
issued by Corporación para el Desarrollo Sostenible del Sur de la Amazonía
(CORPOAMAZONIA) to Universidad de Antioquia. The second collection was made under research permit
IDB0359 issued by Ministerio de medio ambiente y desarrollo sostenible to Universidad de los
Field collections, culture, and maintenance
Six samples were collected in the Chicaque Natural Park, a cloud forest located in the
Municipality of San Antonio del Tequendama, Cundinamarca, Colombia (elev. 1900 to 2700 m,
average temperature of 14ÊC, and an average relative humidity of 85%). The second collection site,
where three samples were collected, was the Amazonian tropical rainforest located in the
Uitoto indigenous community from the Municipality of La Chorrera, Amazonas, Colombia
(elev. 150 m, average temperature of 28ÊC, and an average relative humidity of 95%). The third
2 / 27
location, where one sample was collected in June 2014, was El Amargal Biological Station
Nuqui, a pacific tropical rainforest located in ChocoÂ, Colombia (elev. 40 m, average
temperature of 25ÊC, and an average relative humidity of 95%). The last collection site, where one
sample was collected in November 2014, was in the municipal forest of Mariquita, a lowland
tropical forest located in the inter Andes valley, Tolima, Colombia (elev. 560 m, average
temperature of 28ÊC, and an average relative humidity of 75%). Four additional specimens were
examined from the Herbario Nacional del Ecuador (QCNE), the Herbario de la Universidad de
Antioquia (HUA) and Joao Araujo Personal collection.
The sampling process involved careful examination of the ground to observe the emergence
of stromata from spider cadavers or nests. In the case of spider nests, we extracted the
specimens by digging deep around the site to preserve the complete specimen. Subsequently, a
small piece of tissue from a stroma was submerged and stored in cetyltrimethylammonium
bromide (CTAB) buffer (1.4 M NaCl; 100 mM TriseHCl pH 8.0; 20 mM EDTA pH 8.0; 2%
CTAB w/v) for posterior DNA extraction [
]. The remaining part of the specimen was placed
in a single plastic bag with silica gel for transportation and posterior manipulation in the
In sterile conditions, colonies were obtained by liberating ascospores from a piece (> 3 mm
long) of stroma onto potato dextrose agar (PDA). These were then incubated at 25ÊC [
Sabouraud dextrose agar with 1% (w v-1) yeast extract (SDAY) medium was also used to
maintain isolates. Strains were preserved on filter paper and conserved at 20ÊC [
Specimens and ex-type living cultures were deposited in the collection of the Herbario de
Antioquia (HUA) and the fungal collection of Museo de Historia Natural of Universidad de los
Andes (ANDES-F) in BogotaÂ, Colombia, where they are publicly available (S1 Table).
Collected material was observed under light microscopy after rehydrating in a 3% (w v-1)
potassium hydroxide solution, and then staining with an aqueous solution of Congo red at 5%
(w v-1) and lacto-fuchsine solution (0.1% (w v-1) of fuchsine acid dissolved in lactic acid).
Width and length of perithecia, asci, ascospores, and partspores were measured using a
MOBIC B3000 compound microscope. Methuen Handbook of Color was used for color
descriptions of stromata [
Colony characteristics were described after growing strains on PDA and SDAY for 10 days
at 25ÊC. A piece of mycelium was mounted into microscopic slides and stained with
lactophenol cotton blue dye and lactofuchsine (0.1 g of acid fuchsine in 100 mL of lactic acid). Width
of hyphae, width and length of phialides, and conidia were measured using a MOBIC B3000
and LEICA light microscope. A standard color chart was used to describe the color of colonies
DNA extraction, PCR, and sequencing
The small pieces of fresh tissue immersed in CTAB buffer were ground with sterile plastic
pestles followed by the DNA extraction method described previously [
]. PCR was conducted to
amplify of five nuclear loci, including the small and large subunits (SSU and LSU) of the
nuclear ribosomal DNA (rDNA), the transcription elongation factor-1α (EF-1α), and the first
and second largest subunits of the RNA polymerase II (RPB1 and RPB2). PCR reaction
mixture and PCR program were carried out as previously described [
]. Forward and reverse
nucleotide sequences were assembled and cleaned using Geneious 5.1.2v [
]. Sequences were
manipulated in the software Bioedit Sequence Alingment editor software [
]. New rDNA
sequences generated in this study are publicly available in GenBank (S1 Table).
3 / 27
In order to elucidate the taxonomic status of the fungal material from this study, we
constructed a phylogenetic tree using the nuclear gene regions SSU, LSU, EF-1α, RPB2, and RPB1
from 212 taxa within the Hypocreales following previous studies [24,27±30]. A Maximum
Likelihood analysis was first conducted using RAxML-VI-HPC v 2.0 as part of the CIPRES
]. Evolution model used was GTR-GAMMA with 1,000 bootstrap replicates [
The five nuclear loci were concatenated into a single dataset and eleven data partitions as were
defined previously [
]. Subsequently, a Bayesian Inference analysis was conducted with
MrBayes 3.2.6 and the evolutionary model and the partitions were specified as done for the
RAxML analysis [
]. A total of 10,000,000 MCMCMC generations were performed using a
sample frequency of 500 generations and a burn-in of 25% of the total run. Two runs using
four chains each (one cold and three heated) were performed. Each run was examined with
Tracer 1.5 to verify burn-in parameters and convergence of individual chains [
phylogenetic trees generated in this study are publicly available in TreeBASE (ID: 20481). The final
tree was edited in FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) and Adobe
Illustrator CS5 (Adobe Systems Inc., CA, USA).
Assessment of the metabolic profile
To explore the metabolic composition of the trapdoor spider pathogen, the most recently
collected strain, ANDES-F 1080, was grown on four distinct culture media: i) SDAY as a common
and standard artificial culture medium, ii) SDAY with 5 ppm of sodium selenite (Sigma,
United States) (SDAY-SS) as suggested for the enhancement of nucleoside production [
brown rice (BR) medium as a semi-natural culture medium used for production of biomass
and of reproductive structures in Cordyceps [
], and iv) an integument medium made from
tarantulas (TA) to examine components produced on a substrate similar to that of the host. A
conidial suspension was prepared and adjusted to 105 spores per mL in sterile distilled water
with 0.1% (v v-1) Tween 80. Inoculum was prepared by mixing 1 mL of the conidial
suspension with an equal volume of Sabouraud Dextrose broth with 1% (w v-1) of yeast extract
(SDY), and then incubated at 25ÊC for 3 days. Subsequently, the entire suspension was added
into a 250 mL glass beaker containing 50 mL of each of the four culture media tested. Each
medium with the conidial suspension added, was incubated at 25ÊC for one month with a
photoperiod of 12:12. Colony color and medium color were described as deep orange
(N00A99M80), orange (N00A80M80), or pale orange (N00A50M50), based on the color chart
previously used for cultures. Mycelial density from four-weeks-old fungal cultures was
classified as thin (+), moderate (++), or compact (+++).
All culture media were prepared with 1.5% (w v-1) agar to avoid bias by the presence or
absence of this component. The proportion of brown rice and water used for the BR medium
was 50:60 w v-1. The tarantula integument medium was prepared from adult Theraphosidae
spiders, which were deeply sedated in an ethyl acetate chamber prior to the euthanization as is
suggested by the Institutional Animal Care and Use Committee [
]. Dead specimens were
frozen at -20ÊC, lyophilized, ground, and finally added into the medium in a proportion of 20
Metabolite extraction method
Destructive sampling was used weekly and consisted of a hard separation of all fungal biomass
from each of the culture media. This biomass was kept at -20ÊC until it was lyophilized and
ground for posterior extraction of metabolites. Dry-weight from four-weeks-old fungal
cultures was assessed. Three fractions of extracts deriving from three solvents: n-hexane as a
non4 / 27
polar solvent and methanol and water as polar solvents were included. These fractions were
used for metabolomic profiling and antimicrobial assays. Methanol fractions from one, two,
and three-weeks-old cultures were used to assess antioxidant properties through time.
Methanol and n-hexane extractions were performed as it was described for C. pruinosa
with some modifications [
]. Briefly, the extraction consisted on macerating 100 mg of the
fungal material and adding 2 mL of solvent. Once an emulsion was obtained, samples originating
from methanol and n-hexane fractions were sonicated and then centrifuged at 2,000 rpm for
10 min. The supernatant from these samples was carefully recovered and finally filtered
through a membrane of 0.22 μm. In the case of the water-based extraction, samples were
treated with the ambient temperature water extraction protocol to optimize the extraction of
Metabolomic screen and statistical analyses
Polar and non-polar fractions were lyophilized and dried, respectively, and then adjusted to a
final concentration of 4 mg of extracted fungal biomass per 1 mL of solvent. Five replicates of
these diluted solutions from 12 combined treatments (each culture medium used for fungal
growth and each solvent used for extraction) were analyzed by gas and liquid chromatography
(GC and LC, respectively) coupled to mass spectrometry. All subsequent steps were carried
out at Metabolomic Discoveries GmbH (Potsdam, Germany; http://www.
metabolomicdiscoveries.com/). Derivatization and analyses of metabolites was performed by a
GC-MS 7890A (Agilent, Santa Clara, USA) [
].]. The LC separation was performed using
hydrophilic interaction chromatography with a ZIC-HILIC 3.5 μm, 200 A column (Merck
Sequant, Umeå Sweden), operated by an Agilent 1290 UPLC system (Agilent, Santa Clara,
USA). Acetonitrile was used as the LC mobile phase consisting of a first linear gradient ranging
from 90% to 70% over 15 min, followed by a linear gradient ranging from 70% to 10% over 1
min, a 3 min wash with 10%, and a final re-equilibration wash with 90% for 3 min. The flow
rate was 400 μL min-1 with 1 μL of injection volume. The mass spectrometry was performed
using a high-resolution 6540 QTOF/MS Detector (Agilent, Santa Clara, USA). The measured
metabolite concentration was normalized to internal standard concentrations. Metabolites
were identified by using Metabolomic Discoveries' database entries of authentic standards.
The concentration of metabolites in the different treatments was tested for normality
(Shapiro-Wilk-Test) and variance homogeneity (F-Test) using the appropriate statistical tests
(Students test, Welch test, and Mann-Whitney test). Changes in the concentration of metabolites
among treatments were considered significant with a p-value < 0.05. Venn diagrams and
principal component analyses (PCAs) were used to visualize distinct segregations between
metabolites from the 12 groups studied. Volcano graphs were performed to confirm significant
differences between metabolites from different samples. Statistical analyses were done using
the software JMP Genomics 5.1 (SAS Institute Inc., Cary, NC, 1989±2007). Nucleoside
composition was depicted in pie charts and bar diagrams. An ANOVA test was performed to confirm
differences among mass intensities.
Antimicrobial susceptibility tests
Reference bacterial and fungal strains were obtained from the American Type Culture
Collection and National Collection of Type Cultures. To assess the antimicrobial properties of the
metabolites produced by the fungal strain ANDES-F 1080, nine gram-positive bacteria
(Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis, Kocuria rosea,
Enterococcus faecalis, Enterococcus faecium, Bacillus cereus, Bacillus circulans, and Lysinibacillus
sphaericus), five gram-negative bacteria (Enterobacter aerogenes, Salmonella typhimurium,
5 / 27
Salmonella paratyphi B, Escherichia coli, and Pseudomonas aeruginosa), three molds (Fusarium
oxysporum, Fusarium solani, and Aspergillus fumigatus), and three yeasts (Candida albicans,
Candida parasilopsis, and Candida kruseii) (S2 Table) were included in our study. All strains
were kept at 4ÊC and grown as described below prior to the susceptibility assays. Bacterial
strains were grown in nutrient agar medium at 37ÊC for 24 h, while fungal strains were grown
in SDAY medium at 25ÊC for 5 days.
Bacterial and yeast inoculations were adjusted to a 0.5 McFarland turbidity (1±2 x 106 CFU
mL-1) and spread evenly over the entire surface of the medium using a sterile cotton swab.
Inocula for molds were prepared as described before [38±39]. The susceptibility of the
microorganisms was determined using a well diffusion assay on Mueller-Hinton (MH) agar
(Scharlau, Barcelona, España), following standard procedures as described previously [
originating from four-weeks-old fungal cultures were tested by adding 20 μL of the extract in
each individual well. Methanol fractions were lyophilized and then resuspended on water to
avoid bias by the toxicity of this solvent. Wells with gentamicin (Sigma-Aldrich, St. Louis, MO,
USA), amphotericin (Sigma-Aldrich, St. Louis, MO, USA), and chlorhexidine (Sigma-Aldrich,
St. Louis, MO, USA) at 100 μg mL-1 were used as positive controls for bacteria, molds, and
yeasts, respectively. Wells containing water and n-hexane were used as negative controls.
Microorganisms were incubated under standard conditions as described above. All
experiments were carried out in triplicate. Mean and standard deviation were calculated and used as
the central tendency unit and the statistical dispersion unit, respectively. Zones of growth
inhibition were examined and the diameter of each zone was measured and recorded. Fractions
that showed antimicrobial activity were later tested to determine the Minimal Inhibitory
Concentration (MIC) for each microorganism. The MIC assay was done in triplicate starting with
a concentration of 50 mg of fungal material per 1 mL of solvent and diluting by half the
concentration until an inhibition halo was no longer observed.
Determination of total phenolic content
The total phenolic content (TPC) of methanol fractions was measured using the modified
version of the Folin-Ciocalteu method [
]. The results were estimated using a calibration curve of
gallic acid (Sigma-Aldrich, St. Louis, MO, USA) at a concentration ranging from 100 to
500 ppm; results are expressed as mg of gallic acid equivalents (GAE) per g of dried extract. A
total of eight replicates of each TPC assessment were conducted. Mean and standard deviation
were calculated and used as the central tendency unit and the statistical dispersion unit,
respectively. Differences between data obtained from the same week were compared by conducting a
Student's t-test through the statistical software R v 3.0.1[
]. A p-value < 0.05 was considered
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6 / 27
following digital repositories: PubMed Central (www.ncbi.nlm.nih.gov/pubmed) and LOCKSS
A new fungal pathogen of trapdoor spiders
In this study, 15 specimens were examined from parasitized large spiders belonging to the
suborder Mygalomorpha. Four specimens were found associated with tarantula spiders
(Theraphosidae) and presented a morphology that resembles the concept of Petch from 1933 of
Ophiocordyceps caloceroides [
]. These specimens showed threadlike stromata that were 10±
40 cm long, a fertile area distinguished by its pruinosa appearance due to the large ostioles on
the tiny perithecia (90±250 μm), and short sub-fusiform asci with slightly curved fusiform
multiseptate ascospores. In contrast, the other eleven specimens that were associated to
trapdoor spiders (Idiopidae and Barynchelidae), had slightly capitate stromata that were less than
4 cm long and a fertile area that was cylindrical to clavate. They also had large perithecia (300±
500 μm) with cylindrical asci and filiform ascospores breaking into irregular partspores.
We merged the thirty-three sequences obtained from these specimens with a data set
comprising 227 taxa from six families (Bionectriaceae, Nectriaceae, Hypocreaceae,
Cordycipitaceae, Clavicipitaceae, and Ophiocordycipitaceae) into a unique concatenated alignment of five
loci comprising 4498 base pairs (1115 bp SSU, 977 bp LSU, 1050 bp EF-1α, 1065 bp RPB2, and
791 bp RPB1). Our samples grouped into a new taxon with a strong statistical support
(bootstrap, MLBS = 99; PP = 1) inside the Cordycipitaceae family (Fig 1). They were located in the
same clade as Cordyceps pruinosa and Phytocordyceps ninchukispora, with the specimens of
Ophiocordyceps caloceroides placed as sibling species. Additionally, Cordyceps sp. TL11464 was
placed close to O. caloceroides sharing the same ancestor.
We proposed Cordyceps nidus sp. nov. as a new species of fungal pathogen of trapdoor
spiders (Mygalomorpha: Idiopidae). It is a sibling species of Cordyceps caloceroides, a pathogen of
tarantula spiders (Mygalomorpha: Theraphosidae), which has been classified previously as O.
caloceroides base on asci morphology [
]. Both species, together with C. pruinosa, form a
clade here referred to as KOH+ (Fig 1) due to the shift in color from the typical red
pigmentation to violet when a drop of potassium hydroxide is added.
Cordyceps nidus T. Sanjuan, J.S. ChirivÂõ-SalomoÂn & S. Restrepo sp. nov. Fig 2.
Etymology: In reference to the burrow of the trapdoor spiders.
Description: Stromata claviform, simple, gregarious, fleshy, 10±42 mm long. Fertile part
subcylindrical, apex rounded, pruinosa, coral-red (8B6) to carmine red (9A8) 2.5−18 × 0.5±3
mm. Stipe terete, smooth, brownish red (9C7), 5−34 × 0.5−2 mm. Perithecia
pseudoimmmersed, perpendicular orientation, ellipsoid, 300±500 (-630) × 110±190 (-205) μm (n = 40).
Asci cylindrical, (145-) 190±360 × 2±4 μm (n = 50), cap 3±4.2 × 1.2±3 μm. Ascospores filiform,
hyaline, 100±120 × 1.0 μm (n = 5); breaking irregularly into truncate partspores, (4-)
6±10 × 1μm (n = 50).
Conidial state: Colonies on PDA slow-growing, reaching 28.5±30.0 mm in diameter in 10
days at 25ÊC, white (N00A00M00) to pale orange (N00A50M50), velvety; reverse deep orange
(N00A99M80) to pale orange (N00A50M50), margin entire, soluble pigments not produced.
Colonies on SDAY slow-growing, reaching 20.0−35.0 mm in diameter in 10 days at 25ÊC, pale
orange (N00A50M50), tomentose; reverse deep orange (N00A99M80), margin entire, red
pigments produced on culture media. Fungal odor. Vegetative hyphae 1.0−2.0 μm wide,
smooth7 / 27
Fig 1. Phylogenetic relationships inferred from a Maximum Likelihood analysis of Cordyceps species which parasitize
spiders. Combined data set of SSU, LSU, TEF, RPB1, and RPB2 nuclear loci. Sequences from Verticillium dahlie and Glomerella
cingulata were used as outgroups. Numbers above branches indicate Bootstrap support (BS) and below posterior probability
support in Bayesian analyses (PP). Bionectriaceae, Nectriaceae, Hypocreaceae, Clavicipitaceae, Ophiocordycipitaceae are
collapsed to emphasize of Cordycipitaceae.
8 / 27
Fig 2. Cordyceps nidus. A) Stromata emerging from a nest of trapdoor spider from the family Idiopidae ANDES-F 1080 B) Inside of the nest with the red
stromata on the lid HUA 186125. C) Cross-section of perithecia turned purple with KOH. D) Asci and ascospores stained with fucsin acid dye. E-G)
Conidial state of C. nidus E) Long and slender phialides with a conidial head produces on prostate narrow hypha. F) Conidia in a row showing different
shapes and sizes. G) Synnemata isolated in PDA turned purple on the tip due to the reaction with KOH 3%. H) Synnemata Isolated on brown rice showed
strong red pigments after 30 days. Scale bars: C = 100 μm 10 ×, D-F = 10 μm 100 ×.
walled, regularly septate. Conidiophores arising from submerged hyphae, moderately
branched. Phialides solitary, 5.0−70.0 × 1.0−2.0 μm (n = 30). Conidia forming linear and
globose mucilaginous heads, ellipsoidal to ovoid, usually straight, smooth-walled, 1-celled, 2.0
−7.0 × 1.0−2.0 μm (n = 30).
Host: Stromata emerging from the lid of the burrow of young trapdoor spiders belong to
Idiopidae family (Aranae: Mygalomorpha). White mycelia cover the spider and the burrow
like a net connected to the stromata on the lid.
Specimens examined: COLOMBIA, Cundinamarca, San Antonio del Tequendama,
Chicaque Natural Park, GPS: 4Ê36'28.51@N, 74Ê18'27.50''W, elev. 2,500 m, on the burrow of
Idiopidae spiders. 27 Feb 2011, T. Sanjuan 903, Holotype: HUA 186125.
Additional specimens examined: COLOMBIA, Cundinamarca, San Antonio del
Tequendama, Chicaque Natural Park, GPS: 4Ê36'28.51''N, 74Ê18'27.50''W, elev. 2,500 m. 27 Feb 2011,
T. Sanjuan 904, Isotype: HUA 186186, 10 de Apr de 2011, T sanjuan 930, 12 Nov 2013, T.
Sanjuan 1129, 17 Feb 2015, T. Sanjuan 1163, Paratype: ANDES-F 1080. Amazonas, La Chorrera,
San Francisco Uitoto community, GPS: 1Ê26'58''S, 72Ê47'38''W, elev. 150 m, 11 Sep 2011, T.
Sanjuan 989 Paratype: ANDES-F 1040. Puerto Santander, Araracuara Canyon, GPS: 0Ê37'21''S
9 / 27
Fig 3. Cordyceps caloceroides. A) A long stromata emerging from a big Theraphosidae spider (picture taken by Carlos Padilla on Yasuni Scientific
Research Station, EC). B) Cross section of perithecia stained with Fucsin lactic acid showing the broad ostioles HUA 186166. C) Asci stained with cotton
blue dye showing the curved filiform ascospores Scale bars: A = 5 cm, B = 10 μm 40 ×, C = 10 μm 100 ×.
10 / 27
Tarapaca, El Zafire Reserve, 4Ê0'21'' S, 69Ê53'55'' W, elev. 150 m. 6 Jan 2008, A. Vasco 1962,
HUA 186110. ECUADOR, Orellana, Yasuni National Park. Tiputini Research Station, GPS: 0Ê
38'14.84''S 76Ê09'1.83''W, elev. 200 m, 13 Jul 2004, T. Sanjuan 460, QCNE186272;
Chimborazo, Canton Penipe, Sector Palictahua, 01Ê31'S, 78Ê29' W, elev. 2,400 m, Nov 1 2003, N. Erazo
003, QCNE 186235. BRAZIL, Amazonas, Reserva Adolpho Ducke, 02Ê57'42''S, 59Ê55'40''W,
elev. 100 m. JPM Araujo CALOC.
Host: Stromata emerging from the joins on the body of Tarantula spider belong to
Theraphosidae family (Aranae: Mygalomorpha), located inside of its burrow (Fig 3A)
Note: One additional specimen from Ecuadorian cloud forest which fit the morphology of
C. caloceroides sensu stricto was examined. It was collected in April 2002 by Bryce Kendrick. It
is part of the exhibition of the Vancouver Mycological Society (http://mushroomobserver.org/
Known distribution: tropical rainforest in Bolivia, Brazil, Colombia, Cuba and Ecuador.
Cordyceps nidus produced greater biomass on brown rice culture medium
In general, C. nidus showed the following characteristics: orange (N00A80M80) to white
(N00A00M00) colonies, with a deeper pigmentation on the reverse, a characteristic fungal
smell, and a typical tomentose texture. In spite of the low pigmentation on BR, C. nidus
showed higher values of dry-weight and mycelial density on this medium compared to the
others. The pigmentation on all cultures intensify through time, but pigments on BR intensify less
and were pale orange (N00A50M50) almost all the time. Nevertheless, orange (N00A80M80)
pigments were observed on the reverse of the colony that diffused into the BR medium. Fungal
growth on SDAY and SDAY-SS was similar on all measures recorded. The lowest values of dry
weight for C. nidus were obtained on isolates grown on TA culture medium (Table 1).
The metabolite profile is highly dependent on the medium used to grow the fungus
The metabolic profile of extracts of C. nidus grown on four distinct culture media showed a
high diversity of compounds (S3 Table). Water fractions obtained from all culture media
treatments resulted in a total of 3744 compounds, from which 17.17% of metabolites were
nondependent on the substrate. We also found that BR showed the largest number of unique
compounds (5.07%), followed by TA (2.30%), then SDAY-SS (1.76%), and finally SDAY (0.03%).
On the other hand, methanol fractions summed a total of 3,271 compounds, where 41.18%
were shared among all culture media treatments. In this case, TA produced the greater number
of unique compounds (5.53%), followed by SDAY (3.73%), BR (3.33%), and SDAY-SS
(2.48%). Finally, n-hexane fractions showed a low number of extracted metabolites with a total
SDAY, Sabouraud Dextrose Agar supplemented with yeast extract; SDAY-SS, SDAY supplemented with sodium selenite; BR, brown rice agar; TA,
tarantula integument agar.
11 / 27
of 1,588 compounds. From these extracts, 31.36% of all metabolites were shared among all
treatments. Similar quantities of unique compounds were found in each culture media
treatment when using n-hexane as solvent (S1 Fig).
In addition, the mass intensity of each metabolite was highly distinct. This is directly related
to the concentration of metabolites in each of the extracts. The proportion of metabolites,
especially those that were extracted with methanol, was found to be highly dependent on the
culture medium used to grow C. nidus (Fig 4). Water and n-hexane fractions presented a strong
matrix effect, which is related to high concentration of metabolites, this leads to a lower
detection rate of them. For methanol fractions, the first and second principal components (PC 1
and PC 2) explained 27.45% and 22.5% of the variation, respectively. In the water fractions, PC
1 and PC 2 explained 34.2% and 28.4% of the variation, respectively. For the n-hexane
Fig 4. PCA analysis for the culture medium across different solvent fractions. A) methanol, B) water, and C) n-hexane extractions.
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fractions, no clear differences were observed among culture media, and the combined PCs
only explained 18.5% of the variation.
Semi-natural culture media induced the highest production of nucleosides
The content of nucleosides in C. nidus was compared among the different culture media and
extraction solvents used (Table 2, Fig 5). Certainly, when polar solvents were used for
extraction, a richer set of nucleosides was observed due to their chemical affinity for polar solvents.
Mass intensity values of nucleosides were detected higher in semi-natural culture media (BR
and TA) than in the artificial media tested (SDAY and SDAY-SS). In contrast, the content of
nucleosides of the non-polar fractions was similar among the different culture media used for
fungal growth (Fig 5). Among the nucleosides detected, abacavir, cordycepin, and
N6-hydroxyethyl adenosine predominated with mass intensity values above 106 (Table 2). In water
fractions, the mass intensity value of 5'-Methylthioadenosine also reached this order of
magnitude. Mass intensity values of nucleosides used as markers of quality control of
Cordyceps products were thoroughly studied in polar solvents (Fig 6). Data set from n-hexane
fractions was excluded from this analysis due to its strong matrix effect and the undifferentiated
nucleoside composition (Figs 4 and 5). Remarkably, the mass intensities reported for
cordycepin were the highest reaching values around of 106 (Fig 6B), while the other markers were
reported with mass intensity values around of 104 (Fig 6A and 6C±6E).
These five markers were generally predominant in methanol fractions, especially the mass
intensity of inosine which was always higher independently of the culture medium treatment
in these fractions (Fig 6D). Though matrix effect is also strong for water fractions, the mass
intensity of adenosine, cordycepin, guanosine, and uridine from SDAY-SS treatments were
higher than in methanol ones (Fig 6A±6C and 6E).
Mass intensity values of nucleosides used as markers of quality control of Cordyceps
products were thoroughly studied (Fig 6). Interestingly, mass intensity of cordycepin extracted in
BR was higher in methanol extracts than in water extracts, while the opposite was observed on
TA medium (Fig 6A). In the case of adenosine, inosine, and guanosine, higher concentrations
were found in the methanol extracts obtained from mycelia grown on BR than on the other
culture media tested. Mass intensity values of inosine were two and four times higher with
respect to guanosine and adenosine, respectively (Fig 6B and 6C). Uridine was detected in
higher concentrations in extracts obtained from synthetic culture media (SDAY and
SDAY-SS). Nevertheless, the mass intensity values of uridine compared with the other four
markers were too low to consider it as a quality marker for this species (Fig 6E).
Cordyceps nidus is a constitutive source of potential drugs
The metabolome of C. nidus presented more than 1,000 compounds for each solvent used for
extraction. Several of these metabolites corresponded to peptides, vitamins, organic acids and
sugars (S3 Table), among which some were recognized some of them as potential bioactive
compounds. In Table 3, we depicted the functionality of some metabolites present in methanol
fractions of C. nidus. The metabolites that were selected, were those that were extracted
independently of the substrate used for the fungal growth and that were not commonly observed
or not previously reported in Cordyceps. We also noted that their mass intensities on SDAY
were the smaller ones, which is in concordance with the global diversity of metabolites
extracted. The mass intensities observed for the other substrate treatments showed to be
similar among them.
13 / 27
*Nucleosides detected after dilution steps.
**Nucleoside analogue currently used as pharmaceuticals.
488.94 ± 490.95
0.02 ± 0.04
369.12 ± 825.37
1.12 103 ± 2.51 103
0.04 ± 0.09
806.66 ± 1.80 103
1.38 103 ± 3.09 103
4.57 103 ± 4.86 103
694.29 ± 1.55 103
2.67 103 ± 2.87 103
Methanol fractions showed antibiotic activity against gram-positive bacteria
The MIC for each group of samples is listed in Table 4. This activity was found in the genera
Bacillus, Enterococcus, Kocuria, and Staphylococcus. Enterococcus faecalis and B. circulans
showed the largest inhibition halos (with a MIC of 12.5 mg of fungus per 1 mL of solvent) in
comparison to the gentamicin control. Satellite bacterial colonies were observed inside the
inhibition halos from antimicrobial assays performed with B. circulans. No antimicrobial
activity was present for the water or n-hexane fractions and neither of the extract fractions studied
was able to inhibit growth of molds, yeasts, or gram-negative bacteria.
Cordyceps nidus progressively increased the total phenolic content through time
The TPC of C. nidus samples grown on different culture media through time are shown in
(Fig 7). An exponential increase of TPC is shown for each group of samples on SDAY,
SDAY-SS, and TA through time (R2 > 0.99). On the other hand, a linear increase of TPC was
observed for samples grown on BR medium through time (R2 > 0.99). Fungi grown on SDAY
for four weeks showed the highest TPC levels in comparison with the other treatments (t-test,
p-value<0.05). This phenolic content is followed by that of fungi grown on SDAY-SS and TA
medium, respectively. Brown Rice treatment resulted in the lowest TPC levels at the end of the
evaluated period. These results are in agreement with the pigmentation patterns observed in
16 / 27
Fig 5. Nucleoside composition of from C. nidus grown on different culture media. A) methanol, B)
water, and C) n-hexane extracts.
In this study, the metabolic profile and pharmacological potential of a species of Cordyceps
which parasitizes trapdoor spiders were characterized for the first time. At the same time,
Cordyceps nidus was proposed as a new species closely related to Cordyceps caloceroides, a
recognized tarantula pathogen. Both species were phylogenetically located in the same subclade of
Cordyceps pruinosa, a species studied for its potential pharmacological properties, and were
closer to the medicinale important fungus Cordyceps militaris. Furthermore, the metabolome
of C. nidus has a very diverse composition which is dependent on the substrate where the
fungus is grown. The content of nucleosides and particular metabolites found in C. nidus,
especially when it is cultured on semi-natural culture media, suggests its potential for
pharmaceutical applications. Another possible application relies on the antimicrobial
properties of its metabolites. Methanol fractions presented high activity against gram-positive
bacteria, especially Enterococcus faecalis, which is known to be highly resistant to antibiotics [
We also found the presence of polyphenols in fungal extracts, showing the potential use of C.
nidus as an antioxidant product.
This is the first systematic molecular analysis focusing on the Cordyceps pathogens of large
spiders belonging to the Mygalomorpha subclass. Additionally, we clarified the phylogenetic
position of Ophiocordyceps caloceroides, which was firstly placed in the genus Ophiocordyceps
by Petch in 1933, based on the morphology of its ascospores [
]. Sung et al in 2007, who did
17 / 27
Fig 6. Mass intensities of nucleosides. A) cordycepin, B) adenosine, C) inosine, D) guanosine, and E) uridine extracted from mycelium of C. nidus
grown on different culture media. Methanol (black) and water (white) extracts. Statistical significance for alpha = 0.05: a. 9.26 10−9, b. 1.32 10−7, c.
5.83 10−11, d. 4.11 10−11, e. 3.72 10−3, f. 2.85 10−3, g. 1.64 10−14, h. 2.39 10−12, i. 1.44 10−14.
18 / 27
not include O. caloceroides in their molecular analysis, adopted the same result as well [
However, our specimens exhibited the same morphology of the type material described by
Mains, and the phylogenetic analysis placed them as members of the Cordycipitaceae family
]. Therefore, we returned this taxon to its original position and nomenclature, Cordyceps
caloceroides Berk. and Curt. 1869.
Cordyceps nidus and C. caloceroides were located in the same clade of Cordyceps pruinosa, a
group which is easily recognized by its red pruinose claviform stromata (Fig 2A). The species
in this clade share two non-molecular characters. First, the stromata and mycelia turn violet
when a drop of KOH is added. This biochemical-physiological reaction was firstly reported by
Petch in 1924, when he described C. pruinosa [
]. Also this reaction was reported for
Metarhizium martialis [
]. Second, they are associated with hosts that are enclosed in a structure or
in a habitat with low oxygen concentration: Cordyceps ninchukispora is found in seeds of
Lauraceae plants, C. pruinosa in cocoons of Lepidoptera, Cordyceps sp. TL 11464 in oothecae of
Orthoptera, C. nidus in trapdoor spider nests, and C. caloceroides in deep burrows of
Theraphosidae spiders. This suggests that species from KOH+ clade are a differentiated group,
phylogenetically isolated by the host showing common environmental condition, i.e., low oxygen
concentration. A similar analysis was carried out before recognizing that the species within the
ªravanelliº clade in the Ophiocordycipitaceae family, i.e., Ophiocordyceps heteropoda,
Ophiocordyceps gracillioides, Ophiocordyceps gracillis, Ophiocordyceps blattarioides, and
Ophiocordyceps amazonica, are recognized as phylogenetic species due to their phylogenetic positions and
by their host affiliation and stage of the host, rather than by the slight morphological
differences among them [
]. Once again this confirms that the morphological characters in
Cordyceps s.l. can be acquired or lost through evolution, but the association with a particular host is a
factor strongly correlated with their phylogenetic position.
The intracellular composition of C. nidus is dependent on the substrate used for its culture.
The PCAs based on the culture medium used for fungal growth explained more than 50% of
the variation in the metabolite composition in polar fractions, especially those extracted with
methanol. Among the culture media assayed, BR replicates exhibited the most distinct group
of samples. It is known that the culture medium can induce many changes in the physiology of
a fungus, and these results are in agreement with other studies involving Cordyceps fungi [
Environmental characteristics, such as culture media, pH, and temperature, can be
determining factors in the production of metabolites [57±59]. Further investigation is warranted re the
effect of pH, temperature, and humidity on the production of secondary metabolites,
particularly nucleosides, in cultures of C. nidus. Our results showed that C. nidus produced a high
number of nucleosides, especially in natural culture media, showing its potential use as a
therapeutic agent due to the different biological activities reported for these compounds [
In this study, we focused our analyses on four nucleosides that are used as markers of
quality control in Cordyceps s.l.: cordycepin, adenosine, inosine, and guanosine. The production of
uridine was not significant in any of the four media assessed. As expected, n-hexane was not
effective for nucleosides extraction due to its high polarity. Natural culture media can be used
for growing C. nidus in future studies and for inducing the production of nucleosides. Brown
Rice medium was particularly interesting due to the high fungus biomass production on it.
Curiously, we found that the presence of sodium selenite did not induce the production of
adenosine and cordycepin, which is in disagreement with the promoting activity of selenium
on nucleosides produced by C. militaris [
]. Selenium complexes can promote the activity of
several enzymes involved in cordycepin synthesis [
]. Nevertheless, sodium selenite
composition was adjusted based on this previous work. Thus, an experimental design using higher
compositions may increase the nucleoside production.
19 / 27
Activity modulator of nerve growth
factor, choline acetyltransferase,
and neurotransmitter systems.
Antimicrobial, anti-in¯ammatory, and
Interestingly, some compounds present in C. nidus are related to active compounds
involved in neuronal systems, such as L-acetylcarnitine [43±44], aminocaproic acid [49±52],
], amobarbital [
], and rivastigmine [
]. Among these, L-Acetylcarnitine was
predominant in C. nidus mycelia, reaching mass intensity values up to 107. Furthermore, this
compound was not substrate-dependent. Some Cordyceps spp. were characterized as agents
that prevent neuronal cell death and protective neurons agents [64±66]. These findings suggest
that C. nidus would be a source of compounds for neuronal disease treatments. On the other
hand, we also found several metabolites associated as antimicrobials and antioxidants, which is
in agreement with the tests performed in this study. Remarkably, in this field metabolites such
as cordysinin B are associated with antivirals, which is promising for further studies [
Finally, three nucleoside analogues (Abacavir, Entecavir, and Telbivudine) are important to
mention. Abacavir and Telbivudine are used to prevent and treat hepatitis B infection [67±68],
while Entecavir is a prevention and treatement medication for HIV/AIDS disease [
three metabolites are currently produced by synthetic chemical processes, making C. nidus a
promising natural source of these antiviral compounds.
Strong bactericide activity against highly resistant bacteria, such as E. faecalis, Bacillus spp.,
and Staphylococcus sp., was found in methanol fractions of C. nidus. These results suggest a
novel application against bacteria with high resistance to conventional antibiotics [
Enterococcus is a genus of gram-positive bacteria and some of its species are opportunistic pathogens.
However, these bacteria are more important because they are highly resistant to conventional
antibiotics and present high rates of horizontal gene transfer [
]. Therefore, finding
alternatives to control these bacteria could be of great utility in medicine. On the other hand, the
genus Bacillus is one of the most predominant in the environment. Bacillus spp. are able to
produce endospores and have shown to be resistant to antibiotics [
]. Thus, new alternative
compounds for disease treatments are needed. An important observation was the retarded
growth of B. circulans indicated by the inhibition halos, or the presence of small colonies,
associated with persistence of the bacteria or bacteriostatic activity of the extracts [
In the metabolite profile of the methanol fractions of C. nidus, several chemical components
were found which could potentially act as bactericide. Chloramphenicol, sphingolipids, azoles,
penicillin, and other lactams are some of these compounds present in C. nidus extracts that are
well-documented to have antimicrobial activity [74±77]. Nevertheless,
imidazole-4-acetaldehyde was only present in the methanol fraction, and its concentration was highest when C.
nidus was grown in SDAY and AT media. Given that we found more activity in extracts
20 / 27
derived from these treatments, we suggest that this azole could be a candidate for biological
activity against gram-positive bacteria. On the other hand, the presence of ophiocordin did
not have an effect on tested fungi possibly due to its low concentration in the extracts. Thus,
for future studies, we suggest the performance of antifungal sensitivity tests using
concentrations higher than 50 mg per mL of solvent.
21 / 27
Fig 7. Total phenolic content (TPC) from C. nidus on different culture media. Error bars correspond to the standard deviation
obtained from eight independent replicates.
Polyphenols are important constituents of fungi, given that they can act as antioxidants due
to their capacity to scavenge free radicals [
]. The TPCs of C. nidus cultivated under various
substrates indicate that synthetic culture media are perhaps the best substrates to produce
antioxidants. To further assess the antioxidant properties of C. nidus extracts, antiradical analysis
with 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 2,
2-diphenyl-1-picrylhydrazyl (DPPH) should be performed in the future. Nevertheless, the levels of TPCs on all
treatments are still low in comparison with those obtained from cultures of C. pruinosa, the
closer phylogenetically related species [
]. The optimization of other environmental factors is
needed to induce the production of higher concentrations of this kind of compounds. The
increase of phenols through time is also a remarkable characteristic of this test. A previous
analysis established that the concentration of antioxidants is strongly related to the production
of pigments by the fungus, thus explaining the increase of TPCs through time [
Furthermore, better results for antioxidant production have been obtained by inducing the formation
of fruiting bodies [7±8, 13].
This is the first study that attempts to address the systematics of Cordyceps which parasitize
Mygalomorpha spiders in the Neotropics, and helps to clarify the taxonomy of specimens in
22 / 27
the pictures tagged as Cordyceps (Ophiocordyceps) caloceroides and Cordyceps nidus published
on the internet (e.g. http://mushroomobserver.org/observer/observation_search?pattern=
cordyceps+caloceroides). In the future, a thorough systematic and ecological study will be
necessary to reach a more comprehensive understanding of the Cordyceps-spiders interaction.
This study showed that there are differences in the intracellular metabolite composition of
Cordyceps nidus when grown on different substrates. We also explored the potential of this newly
described pathogen of spiders as a source of biotechnological products. The nucleosides
content and the presence of the particular metabolites described above suggested that this
Cordyceps species may be suitable as a dietary supplement or pharmacological product. Nevertheless,
studies of toxicity and edibility are needed to complement the results obtained in this research.
We also tested the extracts of C. nidus in susceptibility antimicrobial tests and we found
activity against various important bacteria in clinical environments, such as Staphylococcus spp.,
Enterococcus spp., and Bacillus spp., among others. From our assays, we found that methanol
extractions had the highest activity against these pathogens, especially against E. faecalis, which
is highly resistant to antibiotics. Finally, total phenolic content from these extracts showed the
potential of C. nidus as a source of antioxidant products. Nevertheless, we suggested assessing
antiradical activity by stable free radical scavenging analyses with ABTS or DPPH.
S1 Fig. Venn diagram for compounds reported. A) methanol, B) water, and C) n-hexane
S1 Table. GenBank accession numbers for taxa included in this study.
S2 Table. Bacterial and fungal strains used in the antimicrobial susceptibility assays.
S3 Table. Metabolite composition from cultured Cordyceps nidus.
We thank Joao Araujo and Aida Vasco for making collections available for this study. The
photographs ªin situº of Cordyceps nidus were provided by Daniel Winkler (www.
mushroaming.com), and those of Cordyceps caloceroides were taken by Carlos Padilla in
Yasuni Scientific Station, Ecuador (http://www.yasuni.ec). Grateful with Kent Brothers for his
help in the English writing for the paper.
Conceptualization: TS JC SR.
Formal analysis: JC TS NS ST.
Funding acquisition: RS SR.
Investigation: JC TS NS ST.
Project administration: SR RS TS.
Resources: TS RS NS SR.
23 / 27
Supervision: RS SR.
Validation: JC NS ST.
Visualization: JC TS GD SR.
Writing ± original draft: JC GD TS.
Writing ± review & editing: JC TS ST.
24 / 27
25 / 27
Wayne, Pennsylvania 19087 USA: Clinical and Laboratory Standards Institute; 2009. (NCCLS
document M44-A; vol. 29).
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Testing of Filamentous Fungi: Approved Guideline. 1st ed. 950 West Valley Road, Suite 2500, Wayne,
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